Versatile potentiometric metabolite sensing without dioxygen interference

Abstract

The field of electrochemical biosensors has been dominated by amperometric and voltammetric sensors; however, these are limited greatly in their signal dependence on electrode size. Open circuit potentiometric sensors are emerging as an alternative due to their signal insensitivity to electrode size. Here, we present a second-generation biosensor that uses a modified chitosan hydrogel to entrap a dehydrogenase or other oxidoreductase enzyme of interest. The chitosan is modified with a desired electron mediator such that in the presence of the analyte, the enzyme will oxidize or reduce the mediator, thus altering the measured interfacial potential. Using the above design, we demonstrate a swift screening method for appropriate enzyme-mediator pairs based on open circuit potentiometry, as well as the efficacy of the biosensor design using two dehydrogenase enzymes (FADGDH and ADH) and peroxidase. Using 1,2-naphthoquinone as the mediator for FADGDH, dynamic ranges from 0.1 to 50 mM glucose are achieved. We additionally demonstrate the ease of fabrication and modification, a lifetime of ≥28 days, insensitivity to interferents, miniaturization to the microscale, and sensor efficacy in the presence of the enzyme's natural cofactor. These results forge a foundation for the generalized use of potentiometric biosensors for a wide variety of analytes within biologically-relevant systems where oxygen can be an interferent.

Keywords: Alcohol dehydrogenase; FAD-Dependent glucose dehydrogenase; Open circuit potential; Peroxidase; Second-generation biosensor.

Figure 1.

a) Possible crosslinked structures of the three mediators to chitosan using glutaraldehyde in acidic solution, where the possible sites for reaction with glutaraldehyde are shown by red asterisks. b) The reaction that occurs on the surface of the chitosan-mediator-FADGDH carbon electrode to alter the surface potential. Glucose is oxidized to gluconolactone in the presence of the oxidant, which gets reduced to the reductant as a byproduct. The changed [C_R]/[C_O] ratio in the hydrogel then alters the measured open circuit potential.

Figure 2.

The open circuit potential of a chitosan-mediator-FADGDH glassy carbon macroelectrode in (dashed) a blank solution of DPBS and (solid) as glucose is spiked into a solution of DPBS when the mediator is a) 1,2-NQ or b) GCy. Change in the open circuit potential from the initial blank potential as glucose is added to a solution of DPBS when the mediator is c) 1,2-NQ (n = 3) or d) GCy (n = 3). The semi-log plots of parts d-f when the mediator is e) 1,2-NQ (n = 3) or f) GCy (n = 3). The change in open circuit potential of three different chitosan-mediator-FADGDH glassy carbon macroelectrodes as glucose is spiked into a solution of DPBS when the mediator is g) 1,2-NQ or h) GCy.

Figure 3.

a) The change in open circuit potential of a chitosan-mediator-FADGDH glassy carbon macroelectrode in as 5 mM of various sugars are spiked into a solution of DPBS when the mediator is (orange) 1,2-NQ or (blue) GCy (n = 3, each). b) The change open circuit potential of a chitosan-mediator-FADGDH glassy carbon macroelectrode in as 5 mM glucose is spiked into a solution of (solid) DPBS or (checkered) DPBS with 1.3 mM acetaminophen and 170 μM ascorbic acid when the mediator is (orange) 1,2-NQ or (blue) GCy (n = 3, each).

Figure 4.

The change in open circuit potential over 28 days of a chitosan-mediator-FADGDH glassy carbon macroelectrode in DPBS and as 5 mM glucose is spiked into a solution of DPBS when the mediator is a) 1,2-NQ (n = 5) or b) GCy (n = 5).

Figure 5.

The change in open circuit potential for three different chitosan-mediator-FADGDH carbon fiber microelectrodes as glucose is spiked into a solution of DPBS when the mediator is a) 1,2-NQ or b) GCy.

Figure 6.

a) The open circuit potential of a chitosan-GCy-HRP glassy carbon macroelectrode as 30 μM to 50 mM H₂O₂ is spiked into a solution of DPBS (n = 3). b) log scale of part a (n = 3). c) The open circuit potential of a chitosan-GCy-ADH glassy carbon macroelectrode as 0.25 mM to 10 mM NAD⁺ is spiked into a solution of DPBS with 0.15M ethanol (n = 3). d) log scale of part c (n = 3).